Dr Joseph Turner was awarded funding to reduce the reliance on animals in filariasis research, particularly for preclinical testing of therapeutics.
Principal Investigator: Dr Joseph Turner
Organisation: Liverpool School of Tropical Medicine
Award type: PhD studentship
Start date: 2015
Duration: 3 years
Filarial diseases are caused by a group of parasitic worms, known as filariae. Infection occurs when the parasites are transmitted through the bites of flying insects, such as mosquitoes. Conditions are life-long and a major cause of disability, affecting approximately 150 million people worldwide, with a further 1.3 billion at risk of infection. The most wellknown filarial diseases are elephantiasis (lymphatic filariasis) and river blindness (onchocerciasis) which are caused by Brugia malayi and Onchocerca volvulus respectively. Filarial diseases are also of veterinary importance, affecting both cats and dogs.
Mass drug administration community control programmes currently use chemotherapeutic agents, such as ivermectin, to target the transmission stage of the parasitic cycle. However, these are not effective at directly killing the adult parasites and treatment has to be continued annually for five to ten years (depending on the reproductive cycle of the parasite). There is significant investment from governmental and private philanthropic stakeholders to develop curative drugs (macrofilaricides) that eliminate filariasis as a public health problem.
Animals are used for filarial research purposes both to grow the parasites to the life cycle stage of interest (typically the adult stage) and for the testing of potential drugs. The gerbil is the most common model. A major challenge is achieving reproducible levels of adult parasite burden in gerbils because of the high incidence of infection failures and high intra-group variation. For evaluation of the efficacy of an experimental drug, parasites are harvested from donor gerbils, counted and surgically implanted into the peritoneum of recipient animals. There are no biomarkers to assess adult parasite infection status or infectious load and consequently efficacy studies typically involve culling animals at various time points, often up to eight months posttreatment, to quantify the number of parasites. The lack of biomarkers also means animals can develop welfare issues either due to chronic parasitism (e.g. peritoneal adhesions, ascites, adverse behavioural changes) or ageing.
3Rs benefits (actual and potential)
Amy Marriott, the PhD student, has validated the use of immunodeficient inbred mice instead of gerbils to provide a more consistent and reproducible filarial parasite burden for Brugia malayi. This has allowed a reduction in animal numbers from 14 gerbils to eight mice per compound tested (including donor animals and those excluded due to low infection rate).
In the clinic, ultrasonography is used for diagnostic purposes by detecting the random thrashing movement of parasites in the lymphatic system, referred to as the filarial dance sign (FDS). Amy has shown that the FDS can be used in the laboratory to accurately assess the presence or absence of Brugia malayi infection in mice and gerbils (with 100% sensitivity and specificity when more than five worms are present) and also to predict drug effectiveness in a proof-ofprinciple study using known macrofilaricidal and non-macrofilaricidal drugs. Tracking FDS allows longitudinal studies in the same animal avoiding the need for separate groups for experiments with multiple time points.
The use of ultrasonography has led to a further reduction from eight to five mice for drug studies in the Turner laboratory. In addition, implantation surgery can now be avoided in both mice and gerbils as it is possible to inject microscopic larvae intraperitoneally into the experimental animals and track infection by ultrasonography rather than having to take adult parasites from donors. Amy has a first author paper describing the work published in Scientific Reports in 2018.
Scientific and technological benefits
The ultrasonography technique developed by Amy has been adopted in the preclinical validation testing of the candidate macrofilaricide ABBV-4083, in partnership with AbbVie Inc. and the Drugs for Neglected Diseases initiative. The work has been accepted for publication in Science Translational Medicine and the drug candidate has now entered phase II clinical development.
Amy has also optimised a co-culture system of dual human lymphatic endothelial cell monolayers where adult female worms can be maintained with full viability for up to three weeks – three times the duration of commonly used in vitro systems. The longevity of the system allows for long-term and robust in vitro assessment of preclinical therapeutic candidates prior to a decision whether in vivo studies are justified. It has been adopted by Professor Mark Taylor at the Liverpool School of Tropical Medicine in collaboration with Professor David Sattelle at University College London to screen the potency of a new anthelmintic class of molecule against adult stage filariae. Beyond drug screening, the co-culture system can be used to study host/parasite interactions.
The research has been presented at 12 conferences, including at the British Society for Parasitology where Amy was awarded a best poster presentation prize. Joseph is funded as part of the Bill & Melinda Gates Foundation Macrofilaricide Drug Accelerator Consortium and has been able to share the 3Rs advances with academic and industrial partners from Europe and North America. In 2018, Joseph was awarded an NC3Rs project grant of £288k to validate alternative models to cats and dogs for heartworm drug testing. He has established a new collaboration with Bayer Animal Health which will provide veterinary compounds for testing in heartworm drug screening models as part of the project.
This case study was published in our 2019 Research Review.